A rho-associated protein kinase, ROKalpha, binds insulin receptor substrate-1 and modulates insulin signaling.
Insulin receptor substrate-1 (IRS-1) is phosphorylated on multiple tyrosine residues by ligand-activated insulin receptors. These tyrosine phosphorylation sites serve to dock several Src homology 2-containing signaling proteins. In addition, IRS-1 contains a pleckstrin homology domain and a phosphotyrosine binding domain (PTB) implicated in protein-protein and protein-lipid interactions. In a yeast two-hybrid screening using Xenopus IRS-1 (xIRS-1) pleckstrin homology-PTB domains as bait, we identified a Xenopus homolog of Rho-associated kinase alpha (xROKalpha) as a potential xIRS-1-binding protein. The original clone contained the carboxyl terminus of xROKalpha (xROK-C) including the putative Rho binding domain but lacking the amino-terminal kinase domain. Further analyses in yeast indicated that xROK-C bound to the putative PTB domain of xIRS-1. Binding of xROK-C to xIRS-1 was confirmed in Xenopus oocytes after microinjection of mRNA corresponding to xROK-C. Furthermore, microinjection of xROK-C mRNA inhibited insulin-induced mitogen-activated protein kinase activation with a concomitant inhibition of oocyte maturation. In contrast, microinjection of xROK-C mRNA did not inhibit mitogen-activated protein kinase activation or oocyte maturation induced by progesterone or by microinjection of viral Ras (v-Ras) mRNA. These results suggest that xROKalpha may play a role in insulin signaling via a direct interaction with xIRS-1.
Farah
S
S
Ottawa Civic Hospital Loeb Research Institute, Ottawa Civic Hospital, Ottawa K1Y 4E9, Canada.
Agazie
Y
Y
Ohan
N
N
Ngsee
J K
JK
Liu
X J
XJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D029867:Xenopus Proteins; D014982:Xenopus laevis; D054460:rho-Associated Kinases
D018076:DNA, Complementary; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D029867:Xenopus Proteins; C526294:irs1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases